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Oxidative stress has been shown to suppress endothelial nitric oxide synthase expression through activation of the transcription factor fork-head box 0 1 (FOXO1) in cultured endothelial cells. We previously reported that circulating kallistatin levels are markedly reduced in rats with chronic oxidative organ damage. In this study, we investigated the potential role of oxidative stress in suppression of kallistatin expression via FOXO1 activation. In Dahi salt-sensitive (DSS) rats, we found that high salt intake induced a time-dependent correlation of increased thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation) with reduced serum kallistatin levels. Moreover, salt loading provoked an elevation of in situ aortic superoxide formation in association with reduced kallistatin levels. Expression of kallistatin was identified in cultured endothelial cells by immunocytochemistry and flow cytometry; however, H2O2 dose-dependently lowered kallistatin mRNA and protein levels as determined by real-time PCR and Western blot, respectively. Downregulation of kallistatin synthesis by oxidative stress was restored by knockdown of FOXO 1 expression with small-interfering RNA. H2O2 rapidly induced FOXO 1 nuclear translocation, but the effect was blocked by c-Jun NH2-terminal kinase (JNK) inhibitor. Inhibition of JNK by pharmacological inhibitor or small-interfering RNA reversed H2O2's effect on kallistatin expression in endothelial cells. This study demonstrates that an inverse relationship exists between oxidative stress and kallistatin levels in the circulation and blood vessels and that kallistatin expression is negatively regulated by oxidative stress via JNK-dependent FOXO1 activation in cultured endothelial cells. [ABSTRACT FROM AUTHOR]

During exercise, β-feedforward coronary vasodilation has been shown to contribute to the matching of myocardial oxygen supply with the demand of the myocardium. Since both β1- and β2-adrenoceptors are present in the coronary microvasculature, we investigated the relative contribution of these subtypes to β-feedforward coronary vasodilation during exercise as well as to infusion of the β1-agonist norepinephrine and the β1- and β2-agonist isoproterenol. Chronically instrumented swine were studied at rest and during graded treadmill exercise (1-5 km/h) under control conditions and after 3,-blockade with metoprolol (0.5 mg/kg iv) and β1/β2-blockade with propranolol (0.5 mg/kg iv). The selectivity and degree of β-blockade of metoprolol and propranolol were confirmed using isoproterenol infusion (0.05-0.4 μg∙kg-1∙min-1) under resting conditions. Isoproterenol-induced coronary vasodilation was mediated through the β2-adrenoceptor, whereas norepinephrine-induced coronary vasodilation was principally mediated through the β1-adrenoceptor. Exercise resulted in a significant increase in left ventricular norepinephrine release and epinephrine uptake. β2-Adrenoceptor blockade with metoprolol had very little effect under resting conditions. However, during exercise, metoprolol attenuated the increase in myocardial oxygen supply in excess of the reduction in myocardial oxygen demand, as evidenced by a progressive decrease in coronary venous PO2. Consequently, metoprolol caused a clockwise rotation of the relationship between myocardial oxygen consumption and coronary venous PO2. Additional β2-adrenbceptor blockade with propranolol further inhibited myocardial oxygen supply during exercise, resulting in a further clockwise rotation of the relationship between myocardial oxygen consumption and coronary venous PO2. In conclusion, both β1- and β2-adrenoceptors contribute to the β-feedforward coronary resistance vessel dilation during exercise. [ABSTRACT FROM AUTHOR]

Prokineticins are secreted peptides that activate two G protein-coupled receptors: PKR 1 and PKR2. Prokineticins induce angiogenesis and fenestration, but the cognate receptors involved in these functions are unknown. We hypothesized a role for prokineticin receptor signaling pathways and expression profiles in determining the selective effects of prokineticins on coronary endothelial cells (H5V). Activation of the PKR1/MAPK/Akt signaling pathway stimulates proliferation, migration, and angiogenesis in H5V cells, in which PKRI predominates over PKR2. PKR1 was colocalized with Gα11 and was internalized following the stimulation of these cells with prokineticin-2. Knock down of PKR1 or Gα1 expression in H5V cells effectively inhibited prokineticin-2- induced vessel formation and MAPK/Akt activation, indicating a role for PKR1/Gα11 in this process. However, in conditions in which PKR2 predominated over PKR 1, these cells displayed a fenestrated endothelial cell phenotype. H5V cells overexpressing PKR2 displayed large numbers of multivesicular bodies and caveolar clusters and a disruption of the distribution of zonula occluden-1 tight junction protein. Prokineticin-2 induced the colocalization of PKR2 with Gα12, and activated Gα12, which bound to zonula occluden-1 to trigger the degradation of this protein in these cells. Prokineticin-2 induced the formation of vessel-like structures by human aortic endothelial cells expressing only PKR1, and disorganized the tight junctions in human hepatic sinusoidal endothelial cells expressing only PKR2, confirming the divergent roles of these receptors. Our findings show the functional characteristics of coronary endothelial cells depend on the expression of PKR1 and PKR2 levels and the divergent signaling pathways used by these receptors. [ABSTRACT FROM AUTHOR]

Gap junctions are critical to maintaining synchronized impulse propagation and repolarization. Heterogeneous expression of the principal ventricular gap junction protein connexin43 (Cx43) is associated with action potential duration (APD) dispersion across the anterior ventricular wall. Little is known about Cx43 expression patterns and their disparate impact on regional electrophysiology throughout the heart. We aimed to determine whether the anterior and posterior regions of the heart are electro-physiologically distinct. Multisegment, high-resolution optical mapping was performed in canine wedge preparations harvested separately from the anterior left ventricle (aLV; n = 8) and posterior left ventricle (pLy; n = 8). Transmural APD dispersion was significantly greater on the aLV than the pLV (45 ± 13 vs. 26 ± 8.0 ms; P < 0.05). Conduction velocity dispersion was also significantly higher (P < 0.05) across the aLV (39 ± 7%) than the pLV (16 ± 3%). Carbenoxolone perfusion significantly enhanced APD and conduction velocity dispersion on the aLV (by 1.53-fold and 1.36-fold, respectively), but not the pLV (by 1.27-fold and 1.2-fold, respectively), and pro- duced a 4.2-fold increase in susceptibility to inducible arrhythmias in the aLV. Confocal immunofluorescence microscopy revealed significantly (P < 0.05) greater transmural dispersion of Cx43 expression on the aLV (44 ± 10%) compared with the pLV wall (8.3 ± 0.7%), suggesting that regional expression of Cx43 expression patterns may account for regional electrophysiological differences. Computer simulations affirmed that localized uncoupling at the epicardial-midmyocardial interface is sufficient to produce APD gradients observed on the aLV. These data demonstrate that the aLV and pLV differ importantly with respect to their electrophysiological properties and Cx43 expression patterns. Furthermore, local underexpression of Cx43 is closely associated with transmural electrophysiological heterogeneity on the aLV. Therefore, regional and transmural heterogeneous Cx43 expression patterns may be an important mechanism underlying arrhythmia susceptibility, particularly in disease states where gap junction expression is altered. [ABSTRACT FROM AUTHOR]

Although estrogen regulates important aspects of maternal cardiovascular physiology, the role of estrogen on uteroplacental and fetal blood flow is incompletely understood. This study tested the hypothesis that chronically suppressing endogenous estrogen production during the second half of baboon pregnancy alters uterine and fetal blood flow dynamics assessed by ultrasonography. Pregnant baboons were untreated or treated daily with the aromatase inhibitor letrozole or letrozole plus estradiol on days 100-1 60 of gestation (term = 184 days). Blood flow dynamics were determined by Doppler ultrasonography on day 60 and longitudinally between days 110 and 160 of gestation. Letrozole decreased maternal serum estradiol and estrone concentrations by 95% (P < 0.001). Fetal growth biometrical parameters increased (P < 0.001) between days 110 and 160 of gestation and were similar in untreated and letrozole-treated animals. Uterine, umbilical, and fetal middle cerebral artery pulsatility index and resistance index declined (P < 0.01) by 30-50% and uterine artery volume flow increased sixfold (P < 0.001) between days 60 and 160, but values were similar in untreated, letrozole-treated, and letrozole plus estradiol-treated baboons. Thus uterine and fetal artery blood flow indexes, uterine artery volume flow, and fetal growth were maintained at normal levels despite chronic estrogen suppression in the second half of baboon pregnancy. This suggests that elevated levels of endogenous estrogen are not required to maintain low impedance blood flow within the uteroplacental vascular bed during the second half of nonhuman primate pregnancy. [ABSTRACT FROM AUTHOR]

Reduced sarcoplasmic calcium ATPase (SERCA2a) expression has been shown to play a significant role in the cardiac dysfunction in diabetic cardiomyopathy. The mechanism of SERCA2a repression is, however, not known. This study was designed to examine the effect of resveratrol (RSV), a potent activator of SIRT1, on cardiac function and SERCA2a expression in chronic type 1 diabetes. Adult male mice were injected with streptozotocin (STZ) and fed with either a regular diet or a diet enriched with RSV. STZ administration produced progressive decline in cardiac function, associated with markedly reduced SERCA2a and SIRT1 protein levels and increased collagen deposition; RSV treatment to these mice had a tremendous beneficial effect both in terms of improving SERCA2a expression and on cardiac function. In cultured cardiomyo- cytes, RSV restored SERCA2 promoter activity, which was otherwise highly repressed in high-glucose media. Protective effects of RSV were found to be dependent on its ability to activate Silent information regulator (SIRT) 1. In cardiomyocytes, overexpression of SIRT1 was found sufficient to activate SERCA2 promoter in a dose-dependent manner. In contrast, pretreatment of cardiomyocytes with SIRT 1 antag- onist, splitomycin, blocked these beneficial effects of RSV. In addition, SIRT1 knockout (+/-) mice were also found to be more sensitive to STZ-induced decline in SERCA2a rnRNA. The data demonstrate that, in chronic diabetes, 1) the enzymatic activity of cardiac SIRT1 is reduced, which contributes to reduced expression of SERCA2a and 2) through activation of SIRT1, RSV enhances expression of SERCA2a and improves cardiac function. [ABSTRACT FROM AUTHOR]

Recent advances in magnetic resonance (MR) imaging technology have unveiled a wealth of information regarding cardiac histoanatomical complexity. However, methods to faithfully translate this level of fine-scale structural detail into computational whole ventricular models are still in their infancy, and, thus, the relevance of this additional complexity for simulations of cardiac function has yet to be elucidated. Here, we describe the development of a highly detailed finite-element computational model (resolution: ∼125 μm) of rabbit ventricles constructed from highresolution MR data (raw data resolution: 43 X 43 X 36 μm), including the processes of segmentation (using a combination of level-set approaches), identification of relevant anatomical features, mesh generation, and myocyte orientation representation (using a rule-based approach). Full access is provided to the completed model and MR data. Simulation results were compared with those from a simplified model built from the same images but excluding finer anatomical features (vessels/endocardial structures). Initial simulations showed that the presence of trabeculations can provide shortcut paths for excitation, causing regional differences in activation after pacing between models. Endocardial structures gave rise to small-scale virtual electrodes upon the application of external field stimulation, which appeared to protect parts of the endocardium in the complex model from strong polarizations, whereas intramural virtual electrodes caused by blood vessels and extracellular cleft spaces appeared to reduce polarization of the epicardium. Postshock, these differences resulted in the genesis of new excitation wavefronts that were not observed in more simplified models. Furthermore, global differences in the stimulus recovery rates of apexlbase regions were observed, causing differences in the ensuing arrhythmogenic episodes. In conclusion, structurally simplified models are well suited for a large range of cardiac modeling applications. However, important differences are seen when behavior at microscales is relevant, particularly when examining the effects of external electrical stimulation on tissue electrophysiology and arrhythmia induction. This highlights the utility of histoanatomically detailed models for investigations of cardiac function, in particular for future patient-specific modeling. [ABSTRACT FROM AUTHOR]

The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through Zn2+ and glycogen synthase kinase 3β (GSK-3β). Fluorescence dyes including Newport Green Dichiorofluorescein (DCF), 4-amino-5-methylamino2',7'-difluorofluorescein (DAF-FM), and tetramethyirhodamine ethyl ester (TMRE) were used to image free Zn2+ nitric oxide (NO), and mitochondrial membrane potential (ΔΨm), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 mm showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), indicating that morphine mobilizes Zn2+ via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented Zn2+ release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on Zn2+ release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of ΔΨm, indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the Zn2+ chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). The action of morphine on the mPTP was lost in cells transfected with the constitutively active GSK-3β mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK3β. In support, morphine significantly enhanced phosphorylation of GSK-3β at Ser9, and this was blocked by TPEN. GSK-3β small interfering RNA prevented the pore opening in the control cardiomyocytes but failed to enhance the effect of morphine on the mPTP opening. In conclusion, morphine mobilizes intracellular Zn2+ through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3β through Zn2+. [ABSTRACT FROM AUTHOR]

Previously, we showed that sulfaphenazole (SUL), an antimicrobial agent that is a potent inhibitor of cytochrome P4502C9, is protective against ischemia-reperfusion (TIR) injury (Ref. 15). The mechanism, however, underlying this cardioprotection, is largely unknown. With evidence that activation of autophagy is protective against simulated hR in HL-1 cells, and evidence that autophagy is upregulated in preconditioned hearts, we hypothesized that SUL-mediated cardioprotection might resemble ischemic preconditioning with respect to activation of protein kinase C and autophagy. We used the Langendorff model of global ischemia to assess the role of autophagy and protein kinase C in myocardial protection by SUL during hR. We show that SUL enhanced recovery of function, reduced creatine kinase release, decreased infarct size, and induced autophagy. SUL also triggered PKC translocation, whereas inhibition of PKC with chelerythrine blocked the activation of autophagy in adult rat cardiomyocytes. In the Langendorff model, chelerythrine suppressed autophagy and abolished the protection mediated by SUL. SUL increased autophagy in adult rat cardiomyocytes infected with GFP-LC3 adenovirus, in isolated perfused rat hearts, and in mCherry-LC3 transgenic mice. To establish the role of autophagy in cardioprotection, we used the cellpermeable dominant-negative inhibitor of autophagy, Tat-Atg5K130R. Autophagy and cardioprotection were abolished in rat hearts perfused with recombinant Tat-Atg5K130R Taken together, these studies indicate that cardioprotection mediated by SUL involves a PKC-dependent induction of autophagy. The findings suggest that autophagy may be a fundamental process that enhances the heart's tolerance to ischemia. [ABSTRACT FROM AUTHOR]

Ischemia of active skeletal muscle elicits a pressor response termed the muscle metaboreflex. We tested the hypothesis that in normal dogs during dynamic exercise, graded muscle metaboreflex activation (MMA) would progressively attenuate spontaneous heart rate baroreflex sensitivity (SBRS). The animals were chronically instrumented to measure heart rate (HR), cardiac output (CO), mean and systolic arterial pressure (MAP and SAP), and left ventricular systolic pressures (LVSP) at rest and during mild or moderate treadmill exercise before and during progressive MMA [via graded reductions of hindlimb blood flow (HLBF)]. SBRS [slopes of the linear relationships (LRs) between HR and LVSP or SAP during spontaneous sequences of ⩾3 consecutive beats when HR changed inversely vs. pressure] decreased during mild exercise from the resting values (-5.56 ± 0.86 vs. -2.67 ± 0.50 beats·min-1·mmHg-1, P <0.05), and in addition, these LRs were shifted upward. Progressive MMA gradually and linearly increased MAP, CO, and HR; linearly decreased SBRS; and shifted LRs upward and rightward to higher HR and pressures denoting baroreflex resetting. Moderate exercise caused a substantial reduction in SBRS (-1.57 ± 0.38 beats·min-1·mmHg-1,P <0.05) and both an upward and rightward resetting. Gradual MMA at this higher workload also caused significant progressive increases in MAP, CU, and HR and progressive decreases in SBRS, and the LRs were shifted to higher MAP and HR. Our results demonstrate that gradual MMA during mild and moderate dynamic exercise progressively decreases SBRS. In addition, baroreflex control of HR is progressively reset to higher blood pressure and HR in proportion to the extent of MMA. [ABSTRACT FROM AUTHOR]

Activation of the 13-adrenergic receptor (13-AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release from erythrocytes. cAMP levels depend on a balance between synthesis via adenylyl cyclase and hydrolysis by phosphodiesterases (PDEs). Previously, we reported that cAMP increases associated with activation of the β-AR and IPR in rabbit and human erythrocytes are tightly regulated by distinct PDEs (1). Importantly, inhibitors of these PDEs potentiated both increases in cAMP and ATP release. It has been shown that increases in protein kinase (PK) activity can activate PDE3 and PDE4. Both PKA and PKC are present in the erythrocyte and can phosphorylate and activate these PDEs. Here we investigate the hypothesis that PKA regulates PDE activity associated with the β-AR and both PKA and PKC regulate the PDE activity associated with the IPR in rabbit erythrocytes. Pretreatment of erythrocytes with the PKA inhibitor, H89 (10 μ.M), in the presence of the PDE4 inhibitor, rolipram (10 μM), augmented isoproterenol (1 μM)-induced cAMP increases. In contrast, in the presence of the PDE3 inhibitor, cilostazol (10 μM), pretreatment of erythrocytes with either H89 (1 μM) or two chemically dissimilar inhibitors of PKC, calphostin C (1 μM) or GFX109203X (1 μM), potentiated iloprost (1 μM)-induced cAMP increases. Furthermore, pretreatment of erythrocytes with both H89 and GFX109203X in the presence of cilostazol augmented the iloprost-induced increases in cAMP to a greater extent than either PK inhibitor individually. These results support the hypothesis that PDEs associated with receptor-mediated increases in cAMP in rabbit erythrocytes are regulated by kinases specific to the receptor's signaling pathway. [ABSTRACT FROM AUTHOR]

Calcific aortic valve (AV) disease has a high prevalence in the United States, and hypertension is correlated to early onset of the disease. The cause of the disease is poorly understood, although biological and remodeling responses to mechanical forces, such as membrane tension, have been hypothesized to play a role. The mechanical behavior of the native AV has, therefore, been the focus of many recent studies. In the present study, the dynamic deformation characteristics of the AV leaflet and the effects of hypertension on leaflet deformation are quantified. Whole porcine aortic roots were trimmed and mounted in an in vitro pulsatile flow loop and subjected to normal (80/120 mmHg), hypertensive (120/160 mmHg), or severe hypertensive (150/190 mmHg) conditions. Local valve leaflet deformations were calculated with dual-camera photogrammetry method: by tracking the motion of markers placed on the AV leaflets in three dimensions and calculating their spatial deformations. The results demonstrate that, first, during diastole, high transvalvular pressure induces a stretch waveform which plateaus over the diastolic duration in both circumferential and radial directions. During systole, the leaflet stretches in the radial direction due to forward flow drag forces but compresses in the circumferential direction in a manner in agreement with Poisson's effect. Second, average diastolic and systolic stretch ratios were quantified in the radial and circumferential directions in the base and belly region of the leaflet, and diastolic stretch was found to increase with increasing pressure conditions. [ABSTRACT FROM AUTHOR]

This study utilized a transgenic mouse model that expresses an inducible dominant-negative mutation of the transforming growth factor (TGF)-β type II receptor (DnTGFβRII) to define the structural and functional responses of the left ventricle (LV) to pressure-overload stress in the absence of an intact TGF-β signaling cascade. DnTGFβRII and nontransgenic (NTG) control mice (male, 8-10 wk) were randomized to receive Zn2+ (25 mM ZnSO4 in drinking H2O to induce DnTGFβRII gene expression) or control tap H2O and then further randomized to undergo transverse aortic constriction (TAC) or sham surgery. At 7 days post-TAC, interstitial nonmyocyte proliferation (Ki67 staining) was greatly reduced in LV of DnTGFβRll+Zn2+ mice compared with the other TAC groups. At 28 and 120 days post-TAC, collagen deposition (picrosirius-red staining) in LV was attenuated in DnTGFβRll+Zn2+ mice compared with the other TAC groups. LV end systolic diameter and end systolic and end diastolic volumes were markedly increased, while ejection fraction and fractional shortening were significantly decreased in TAC-DnTGFβRll+Zn2+ mice cornpared with the other groups at 120 days post-TAC. These data indicate that interruption of TGF-β signaling attenuates pressure-overload-induced interstitial nonmyocyte proliferation and collagen deposition and promotes LV dilation and dysfunction in the pressure-overloaded heart, thus creating a novel model of dilated cardiomyopathy. [ABSTRACT FROM AUTHOR]

The Na+/Ca2+ exchanger (NCX) is proposed to be an important protein in the regulation of Ca2+ movements in the heart. This Ca2+ regulatory action is thought to modulate contractile activity in the heart under normal physiological conditions and may contribute to the Ca2+ overload that occurs during ischemic reperfusion challenge. To evaluate these hypotheses, adult rat cardiomyocytes were exposed to an adenovirus that codes for short hairpin RNA (shRNA) targeting NCX gene expression through RNA interference. An adenovirus transcribing a short RNA with a scrambled nucleotide sequence was compared with the NCX-shRNA nucleotide sequence and used as a control. Freshly isolated rat cardiomyocytes were infected with virus for 48 h before examination. Cardiomyocytes maintained their characteristic morphological appearance during this short time period after isolation. NCX expression was inhibited by up to ∼60% by the shRNA treatment as determined by Western blot analysis. The depletion in NCX protein was accompanied by a significant depression of NCX activity in shRNA-treated cells. Ca2+ homeostasis was unaltered in the shRNA-treated cells upon electrical stimulation compared with control cells. However, when cardiomyocytes were exposed to a simulated ischemic solution, NCX-depleted cells were significantly protected from the rise in cytoplasmic Ca2+ and damage that was detected in control cells during ischemia and reperfusion. Our data support the role for NCX in ischemic injury to the heart and demonstrate the usefulness of altering gene expression with an adenoviral-delivery system of shRNA in adult cardiomyocytes. [ABSTRACT FROM AUTHOR]

To simulate the effects of shear stress in regions of the vasculature prone to developing atherosclerosis, we subjected human umbilical vein endothelial cells to reversing shear stress to mimic the hemodynamic conditions at the wall of the carotid sinus, a site of complex, reversing blood flow and commonly observed atherosclerosis. We compared the effects of reversing shear stress (time-average: 1 dyn/cm2, maximum: + 11 dyn/cm2, minimum: -11 dyn/cm2, 1 Hz), arterial steady shear stress (15 dyn/cm2), and low steady shear stress (1 dyn/cm2) on gene expression, cell proliferation, and monocyte adhesiveness. Microarray analysis revealed that most differentially expressed genes were similarly regulated by all three shear stress regimens compared with static culture. Comparisons of the three shear stress regimens to each other identified 138 genes regulated by low average shear stress and 22 genes regulated by fluid reversal. Low average shear stress induced increased cell proliferation compared with high shear stress. Only reversing shear stress exposure induced monocyte adhesion. The adhesion of monocytes was partially inhibited by the incubation of endothelial cells with ICAM-1 blocking antibody. Increased heparan sulfate proteoglycan expression was observed on the surface of cells exposed to reversing shear stress. Heparinase III treatment significantly reduced monocyte adhesion. Our results suggest that low steady shear stress is the major impetus for differential gene expression and cell proliferation, whereas reversing flow regulates monocyte adhesion. [ABSTRACT FROM AUTHOR]

Inorganic nitrite has recently been recognized to possess vascular activity that is enhanced in hypoxia. This has been demonstrated in humans in the forearm vascular bed. In animal models nitrite reduces pulmonary vascular resistance, but its effects upon the pulmonary circulation of humans have not yet been demonstrated. This paradigm is of particular interest mechanistically since the pulmonary vasculature is known to behave differently to the systemic. To investigate, 18 healthy volunteers were studied in a hypoxic chamber (inspired oxygen, 12%) or while breathing room air. Each received an infusion of sodium nitrite (1 μmol/min) or 0.9% saline. Three protocols were performed: nitrite! hypoxia (n = 12), saline/hypoxia (n = 6), and nitrite/normoxia (n = 6). Venous blood was sampled for plasma nitrite, forearm blood flow was measured by strain-gauge plethysmography, and pulmonary arterial pressure was measured by transthoracic echocardiography. Plasma nitrite doubled and clearance kinetics were similar whether nitrite was infused in hypoxia or normoxia. During hypoxia, nitrite increased forearm blood flow (+ 36%, P < 0.001) and reduced three separate indirect indexes of pulmonary arterial pressure by 16%, 12%, and 17% (P < 0.01). Pulmonary, but not systemic, arterial effects persisted 1 h after stopping the infusion, at a time when plasma nitrite had returned to baseline. No effects were observed during normoxia. Therefore, in hypoxic but not normoxic subjects, sodium nitrite causes arterial and pulmonary vasodilatation. In addition, hypoxia-induced pulmonary vasoconstriction was attenuated for a prolonged period and not dependent on a simultaneous elevation of plasma nitrite. This finding is consistent with the direct extravascular metabolism of nitrite to nitric oxide to effect hypoxia-associated bioactivity. [ABSTRACT FROM AUTHOR]

The atrioventricular (AV) node filters atrial impulses. Underlying rate-dependent refractory properties are assessed with the effective (ERPN; longest nonconducted atrial cycle length) and functional (FRPN; shortest His bundle cycle) refractory period determined with premature protocols at different basic rates. Fast rates prolong ERPN and shorten FRPN, but these effects vary with subjects, age, and species. We propose that these opposite and variable effects reflect the net sum of concurrent cumulative and noncumulative effects associated with basic (BCL) and pretest cycle length (PTCL), respectively. To test this hypothesis, we assessed selecfive and combined effects of five BCL (S1S1) and six PTCL (S1S2) on ERPN, FRPN, and their subintervals (ERPN = A2H2 + H2A3 and FRPN = H2A3 + A3H3, where A is atrium and H is His bundle) with S1S2S3 protocols in six rabbit heart preparations. At control BCL, PTCL shorteningsprolongedERPN(113 ± 12 vs. 101 ± 14ms,P<0.0l)as a net result of prolonged A2H2 and curtailed H2A3. At control PTCL, BCL shortenings increased ERPN (127 ± 20 vs. 101 ± 14 ms, P < 0.01) by prolonging A2H2. FRPN did not vary with BCL but decreased (163 ± 6 vs. 175 ± 10 ms,P < 0.01) with PTCL that curtailed H2A3. Equal BCL and PTCL shortenings as in standard protocols prolonged ERPN but left FRPN unchanged. Notably, ERPN and FRPN significantly correlated through their H2A3 subinterval. In conclusion, BCL and PTCL are both important determinants of AV nodal refractoriness and together account for rate-induced changes in ERPN and FRPN observed during standard premature protocols. ERPN and FRPN are related variables. Similar functional rules may govern nodal refractory behavior during supraventricular tachyarrhythmias. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy represents a severe inherited disease of striated muscle. It is caused by a mutation of the dystrophin gene and characterized by a progressive loss of skeletal muscle function. Most patients also develop a dystrophic cardiomyopathy, resulting in dilated hypertrophy and heart failure, but the cellular mechanisms leading to the deterioration of cardiac function remain elusive. In the present study, we tested whether defective excitation-contraction (E-C) coupling contributes to impaired cardiac performance. "E-C coupling gain" was determined in cardiomyocytes from control and dystrophin-deficient mdx mice. To this end, L-type Ca2+ currents (ICaL) were measured with the whole cell patch-clamp technique, whereas Ca2+ transients were simultaneously recorded with confocal imaging of fluo-3. Initial findings indicated subtle changes of E-C coupling in mdx cells despite matched Ca2+ loading of the sarcoplasmic reticulum (SR). However, lowering the extracellular Ca2+ concentration, a maneuver used to unmask latent E-C coupling problems, was surprisingly much better tolerated by mdx myocytes, suggesting a hypersensitive E-C coupling mechanism. Challenging the SR Ca2+ release by slow elevations of the intracellular Ca2+ concentration resulted in Ca2+ oscillations after a much shorter delay in mdx cells. This is consistent with an enhanced Ca2+ sensitivity of the SR Ca2+-release channels [ryanodine receptors (RyRs)I. The hypersensitivity could be normalized by the introduction of reducing agents, indicating that the elevated cellular ROS generation in dystrophy underlies the abnormal RyR sensitivity and hypersensitive E-C coupling. Our data suggest that in dystrophin-deficient cardiomyocytes, E-C coupling is altered due to potentially arrhythmogenic changes in the Ca2+ sensitivity of redox-modified RyRs. [ABSTRACT FROM AUTHOR]

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demon- strate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ. [ABSTRACT FROM AUTHOR]

We sought to examine the potential role of oxidative stress on skeletal muscle function with advancing age. Nuclear magnetic resonance (NMR) was employed to simultaneously assess muscle perfusion (arterial spin labeling) and energetics (3 `P NMR spectroscopy) in the lower leg of young (26 ± 5 yr, n = 6) and older (70 ± 5 yr, n = 6) healthy volunteers following the consumption of either placebo (PL) or an oral antioxidant (AO) cocktail (vitamins C and E and x-lipoic acid), previously documented to decrease plasma free radical concentration. NMR measurements were made during and after 5 mm of moderate intensity (5 W) plantar flexion exercise. AO administration significantly improved end-exercise perfusion (AO, 50 ± 5, and PL, 43 ± 4 ml∙100 g-1∙min-1) and postexercise perfusion area under the curve (AO, 1,286 ± 236, and PL, 866 ± 144 ml/100 g) in older subjects, whereas AO administration did not alter hemodynamics in the young group. Concomitantly, muscle oxidative capacity (time constant of phosphocreatine recovery, `r) was improved following AO in the older (AO, 43 ± 1, and PL, 51 ± 7 s) but not the young (AO, 54 ± 5, and PL, 48 ± 7 s) group. These findings support the concept that oxidative stress may be partially responsible for the age-related decline in skeletal muscle perfusion during physical activity and reveal a muscle metabolic reserve capacity in the elderly that is accessible under conditions of improved perfusion. [ABSTRACT FROM AUTHOR]

Our previous study showed that stimulation of adenosine A1 receptors located in the nucleus of the solitary tract (NTS) exerts counteracting effects on the iliac vascular bed: activation of the adrenal medulla and 3-adrenergic vasodilation versus vasoconstriction mediated by neural and unknown humoral factors. In the present study we investigated the relative contribution of three major potential humoral vasoconstrictors: vasopressin, angiotensin II, and norepinephrine in this response. In urethane-chioralose anesthetized rats we compared the integral changes in iliac vascular conductance evoked by microinjections into the NTS of the selective A1 receptor agonist N6-cyclopentyladenosine (CPA; 330 pmol in 50 nI) in intact (Int) animals and following: V1 vasopressin receptor blockade (VX), angiotensin II AT1 receptor blockade (ATX), bilateral adrenalectomy + ganglionic blockade (ADX + GX; which eliminated the potential increases in circulating norepinephrine and epinephrine), ADX + OX + VX and ADX + OX + VX + ATX. In mt animals, stimulation of NTS A1 adenosine receptors evoked typical variable responses with prevailing pressor and vasoconstrictor effects. VX reversed the responses to depressor ones. ATX did not significantly alter the responses. ADX + GX accentuated pressor and vasoconstrictor responses, whereas ADX + OX + VX and ADX + OX + VX + ATX virtually abolished the responses. Stimulation of NTS A1 adenosine receptors increased circulating vasopressin over fourfold (26.4 ± 10.4 vs. 117.0 ± 19 pg/mI). These data strongly suggest that vasopressin is a major vasoconstrictor factor opposing 3-adrenergic vasodilation in iliac vascular responses triggered by stimulation of NTS A1 adenosine receptors, whereas angiotensin II and norepinephrine do not contribute significantly to the vasoconstrictor responses. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF)-α is a proinflammatory cytokine that has been implicated in the pathogenesis of heart failure. In contrast, we have recently shown that myocardial levels of TNF-α are acutely elevated in the aortocaval (AV) fistula model of heart failure. Based on these observations, we hypothesized that progression of adverse myocardial remodeling secondary to volume overload would be prevented by inhibition of TNF-α with etanercept. Furthermore, a principal objective of this study was to elucidate the effect of TNF-α inhibition during different phases of the myocardial remodeling process. Eight-week-old male Sprague-Dawley rats were randomly divided into the following three groups: sham-operated controls, untreated AV fistulas, and etanercept-treated AV fistulas. Each group was further subdivided to study three different time points consisting of 3 days, 3 wk, and 8 wk postfistula. Etanercept was administered subcutaneously at 1 mg/kg body wt. Etanercept prevented collagen degradation at 3 days and significantly attenuated the decrease in collagen at 8 wk postfistula. Although TNF-α antagonism did not prevent the initial ventricular dilatation at 3 wk postfistula, etanercept was effective at significantly attenuating the subsequent ventricular hypertrophy, dilatation, and increased compliance at 8 wk postfistula. These positive adaptations achieved with etanercept administration translated into significant functional improvements. At a cellular level, etanercept also markedly attenuated increases in cardiomyocyte length, width, and area at 8 wk postfistula. These observations demonstrate that TNF-α has a pivotal role in adverse myocardial remodeling and that treatment with etanercept can attenuate the progression to heart failure. [ABSTRACT FROM AUTHOR]

Neointima formation often occurs in regions where the endothelium has been damaged and the transmural interstitial flow is elevated. Vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) contribute to intimal thickening by migrating from the media and adventitia into the site of injury. In this study, for the first time, the direct effects of interstitial flow on SMC and FB/MFB migration were investigated in an in vitro three-dimensional system. Collagen I gels were used to mimic three-dimensional extracellular matrix (ECM) for rat aortic SMCs and FBs/MFBs. Exposure to interstitial flow induced by 1 cmH2O pressure differential (shear stress, ∼0.05 dyn/cm2 flow velocity, ∼0.5 p.m/s; and Darcy permeability, ∼10-11 cm2) substantially enhanced cell motility. Matrix metalloproteinase (MMP) inhibitor (GM-6001) abolished flow-induced migration augmentation, which suggested that the enhanced motility was MMP dependent. The upregulation of MMP-1 played a critical role for the flow-enhanced motility, which was further confirmed by silencing MMP-1 gene expression. Longer exposures to higher flows suppressed the number of migrated cells, although MMP-1 gene expression remained high. This suppression was a result of both flow-induced tissue inhibitor of metalloproteinase-1 upregulation and increased apoptotic and necrotic cell death. Interstitial flow did not affect MMP-2 gene expression or activity in the collagen I gel for any cell type. Our findings shed light on the mechanism by which vascular SMCs and FBs/MFBs contribute to intimal thickening in regions of vascular injury where interstitial flow is elevated. [ABSTRACT FROM AUTHOR]

The SCN5A-encoded cardiac sodium channel underlies excitability in the heart, and dysfunction of sodium current (INa) can cause fatal ventricular arrhythmia in maladies such as long QT syndrome, Brugada syndrome (BrS), and sudden infant death syndrome (SIDS). The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1), but the function for this enzyme is unknown. Mutations in GPD1L have been associated with BrS and SIDS and decrease INa through an unknown mechanism. Using a heterologous expression system, we show that GPD1L associated with SCN5A and that the BrSand SIDS-related mutations in GPD1L caused a loss of enzymatic function resulting in glycerol-3-phosphate PKC-dependent phosphorylation of SCN5A at serine 1503 (S1503) through a GPD1L-dependent pathway. The direct phosphorylation of S1503 markedly decreased INa. These results show a function for GPD1L in cell physiology and a mechanism linking mutations in GPD1L to sudden cardiac arrest. Because the enzymatic step catalyzed by GPD1L depends upon nicotinamide adenine dinucleotide, this GPD1L pathway links the metabolic state of the cell to INa and excitability and may be important more generally in cardiac ischemia and heart failure. [ABSTRACT FROM AUTHOR]

The discovery that hemoglobin, albumin, and glutathione carry and release nitric oxide (NO) may have consequences for movement of NO by blood within microvessels. We hypothesize that NO in plasma or bound to proteins likely survives to downstream locations. To confirm this hypothesis, there must be a finite NO concentration ([NO]) in arteriolar blood, and upstream resistance vessels must be able to increase the vessel wall [NO] of downstream arterioles. Arteriolar blood NO was measured with NO-sensitive microelectrodes, and vessel wall [NO] was consistently 25-40% higher than blood [NO]. Localized suppression of NO production in large arterioles over 500-1,000 μm with L-nitroarginine reduced the [NO] ∼40%, indicating as much as 60% of the wall NO was from blood transfer. Flow in mesenteric arteries was elevated by occlusion of adjacent arteries to induce a flow-mediated increase in arterial NO production. Both arterial wall and downstream arteriolar [NO] increased and the arterioles dilated as the blood [NO] was increased. To study receptor-mediated NO generation, bradykinin was locally applied to upstream large arterioles and NO measured there and in downstream arterioles. At both sites, [NO] increased and both sets of vessels dilated. When isoproterenol was applied to the upstream vessels, they dilated, but neither the [NO] or diameter downstream arterioles increased. These observations indicate that NO can move in blood from upstream to downstream resistance vessels. This mechanism allows larger vessels that generate large [NO] to influence vascular tone in downstream vessels in response to both flow and receptor stimuli. [ABSTRACT FROM AUTHOR]

Diastolic depolarization (DD) of atrial myocytes can lead to spontaneous action potentials (APs) and, potentially, atrial tachyarrhythmias. This study examined the hypotheses that 1) a slowly inactivating component of the Na+ current (referred to as late INa) may contribute to DD and initiate AP firing and that 2) blocking lateINa will reduce spontaneous and induced firing of APs by atrial myocytes. Guinea pig atrial myocytes without or with DD and spontaneous AP firing were studied using the whole cell patch-clamp technique. In experiments using cells with a stable resting membrane potential (no spontaneous DD or firing), hydrogen peroxide (H2O2, 50 μmol/I) caused DD and AP firing. The H2O2-induced activity was suppressed by the late INa inhibitors tetrodotoxin (TFX, 1 μmol/l) and ranolazine (5 μmol/l). In cells with DD but no spontaneous APs, the late INa enhancer anemone toxin II (ATX-II, 10 nmol/l) accelerated DD and induced APs. In cells with DD and spontaneous AP firing, TTX and ranolazine (both, 1 μmol/l) significantly reduced the slope of DD by 81 ± 12% and 75 ± 11% and the frequency of spontaneous firing by 70 ± 15% and 74 ± 9%, respectively. Ramp voltage-clamp simulating DD elicited a slow inward current. TTX at 1, 3, and 10 p.mol/l inhibited this current by 41 ± 4%, 73 ± 2%, and 91 ± 1%, respectively, suggesting that a slowly inactivating `Na underlies the DD. ATX-II and H2O2 increased the amplitude of this current, and the effects of ATX-II and H2O2 were attenuated by ranolazine or TTX. In conclusion, late INa can contribute to the DD of atrial myocytes and the inhibition of this current suppresses atrial DD and spontaneous APs. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) has anti-inflammatory properties. We previously reported that acute treatments with inhaled CO inhibit vascular inflammation and hypoxia-induced vasoocciusion in sickle cell disease mouse models. Therefore, we hypothesized that chronic CO inhalation would decrease vascular inflammation and organ pathology in a sickle cell disease mouse model. The treatment of sickle cell disease mice with 25 or 250 parts/million inhaled CO for I h/day, 3 days/wk for 8-10 wk significantly decreased the total mean white blood cell, neutrophil, and lymphocyte counts in peripheral blood. Eight weeks of 250 parts/million CO treatments reduced staining for myeloid and lymphoid markers in the bone marrow of sickle mice. Bone marrow from treated sickle mice exhibited a significant decrease in colony-forming unit granulocyte-macrophage during colony-forming cell assays. Anti-inflammatory signaling pathways phospho-Akt and phospho-p38 MAPK were markedly increased in CO-treated sickle livers. Importantly, CO-treated sickle mice had a significant reduction in liver parenchymal necrosis, reflecting the anti-inflammatory benefits of CO. We conclude that inhaled CO may be a beneficial anti-inflammatory therapy for sickle cell disease. [ABSTRACT FROM AUTHOR]

Voltage-dependent calcium (Ca2+, Cav1.2) channels are the primary Ca2+ entry pathway in smooth muscle cells of resistance-size (myogenic) arteries, but their molecular identity remains unclear. Here we identified and quantified Cav1.2 α1-subunit splice variation in myocytes of rat resistance-size (100-200 urn diameter) cerebral arteries. Full-length clones containing either exon 1b or the recently identified exon Ic exhibited additional primary splice variation at exons 9*, 21/22, 31/32, and ± 33. Real-time PCR confirmed the findings from full-length clones and indicated that the major Cav1.2 variant contained exons 1c, 8, 21, and 32+33, with ∼57% containing 9*. Exon 9* was more prevalent in clones containing 1c (72%) than in those containing 1b (33%), suggesting exon-selective combinatorial splicing. To examine the functional significance of this splicing profile, membrane currents produced by each of the four exon lb/cl ± 9* variants were characterized following transfection in HEK293 cells. Exon Ic and 9* caused similar hyperpolarizing shifts in both current-voltage relationships and voltage-dependent activation of currents. Furthermore, exon 9* induced a hyperpolarizing shift only in the voltage-dependent activation of channels containing exon ib, but not in those containing exon 1c. In contrast, exon 1b, 1c, or +9* did not alter voltagedependent inactivation. In summary, we have identified the Cav1.2 a,-subunit splice variant population that is expressed in myocytes of resistance-size arteries and the unique electrophysiological properties of recombinant channels formed by exon 1 and 9* variation. The predominance of exon 1c and 9* in smooth muscle cell Cavl.2 channels causes a hyperpolarizing shift in the voltage sensitivity of currents toward the physiological arterial voltage range. [ABSTRACT FROM AUTHOR]

The objective of this study was to better understand the role of transforming growth factor-β (TGF-β) and its primary signaling protein Smad3 in the development of intimal hyperplasia. Male Sprague-Dawley rats underwent left carotid balloon injury followed by intra-arterial infection with adenovirus-expressing Smad3 (AdSmad3). In uninfected injured arteries, endogenous Smad3 was upregulated with the expression peaking at 14 days. Moreover, in arteries infected with AdSmad3, we observed an enhancement of intimal hyperplasia and increased vascular smooth muscle cell (VSMC) proliferation. The novel finding, that TGF-β/Smad3 stimulated rather than inhibited VSMC proliferation, was confirmed in cultured VSMCs infected with AdSmad3 and treated with TGF-β. To identify the mechanism underlying TGF-β/ Smad3-mediated VSMC proliferation, we studied the cyclin-dependent kinase inhibitor p27. Although the upregulation of Smad3 in VSMCs had no significant effect on total p27 levels, Smad3 did stimulate the phosphorylation of p27 at serine-10 as well as the nuclear export of p27, events associated with cell proliferation. Furthermore, serine-10-phosphorylated p27 was also increased in AdSmad3-infected injured rat carotid arteries, demonstrating the existence of this same mechanism in vivo. In conclusion, our findings identify a novel mechanism for the effect of TGF-β on intimal hyperplasia. In the presence of elevated levels of Smad3 that develop in response to injury, TGF-β stimulates smooth muscle cell proliferation through a mechanism involving the phosphorylation and nuclear export of p17. [ABSTRACT FROM AUTHOR]

Actin polymerization has recently emerged as an important cellular process that regulates smooth muscle contraction. Abelson tyrosine kinase (Abl) has been implicated in the regulation of actin dynamics and force development in vascular smooth muscle. In the present study, the systolic blood pressure was lower in Abl-/- knockout mice compared with wild-type mice. The knockout of Abi diminished the tyrosine phosphorylation of p130 Crk-associated substrate (CAS, an adapter protein associated with smooth muscle contraction) in resistance arteries upon stimulation with phenylephrine or angiotensin II. The agonist-elicited enhancement of F-actin-to-G-actin ratios in arteries assessed by fluorescent microscopy was also reduced in Abl-/- mice. It has been known that vinculin is a structural protein that links actin filaments to extracellular matrix via transmembrane integrins, whereas paxillin is a signaling protein associated with focal contacts mediating actin cytoskeleton remodeling. The expression of vinculin and paxillin at protein and messenger levels was lower in arterial vessels from AbI knockout mice. However, the agonist-induced increase in myosin phosphorylation was not attenuated in arteries from AbI knockout mice. These results indicate that AbI differentially regulates Crk-associated substrate, vinculin, and paxillin in arterial vessels. The Abl-regulated cellular process and blood pressure are independent of myosin activation in vascular smooth muscle. [ABSTRACT FROM AUTHOR]

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chioroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated ct-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics. [ABSTRACT FROM AUTHOR]

B-type natriuretic peptide (BNP) is a peptide hormone of myocardial origin with significant cardioprotective properties. Patients with myocardial ischemia present with high levels of BNP in plasma and elevated expression in the myocardium. However, the molecular mechanisms of BNP induction in the ischemic myocardium are not well understood. The aim of the investigation was to assess whether myocardial hypoxia induces the production of BNP in human ventricular myocytes. To test the hypothesis that reduced oxygen tension can directly stimulate BNP gene expression and release in the absence of hemodynamic or neurohormonal stimuli, we used an in vitro model system of cultured human ventricular myocytes (ACI6 cells). Cells were cultured under normoxic (21% O2) or hypoxic (5% O2) conditions for up to 48 h. The accumulation of BNP, atrial natriuretic peptide (ANP), and vascular endothelial growth factor (VEGF) was then measured. Hypoxia stimulated the protein release of BNP and VEGF but not ANP. In concordance, the increased mRNA levels of BNP and VEGF but not ANP were found on culturing AC16 cells under hypoxic conditions. The analysis of the transcriptional activity of the hypoxia-inducible factor 1 (HIF-1) in nuclear extracts showed that HIF1 activity was induced under hypoxic conditions. Finally, the treatment of AC16 cells with the HIF-1 inhibitor rotenone in hypoxia inhibited BNP and VEGF release. In conclusion, these data indicate that hypoxia induces the synthesis and secretion of BNP in human ventricular myocytes, likely through HIF-1-enhanced transcriptional activity. [ABSTRACT FROM AUTHOR]

Given that the physiology of heme oxygenase-1 (HO-1) encompasses mitochondrial biogenesis, we tested the hypothesis that the HO-1 product, carbon monoxide (CO), activates mitochondnal biogenesis in skeletal muscle and enhances maximal oxygen uptake (VO2max) in humans. In 10 healthy subjects, we biopsied the vastus lateralis and performed VO2max tests followed by blinded randomization to air or CO breathing (1 h/day at 100 parts/million for 5 days), a contralateral muscle biopsy on day 5, and repeat VO2max testing on day 8. Six independent subjects underwent CO breathing and two muscle biopsies without exercise testing. Molecular studies were performed by real-time RT-PCR, Western blot analysis, and immunochemistry. After VO2max testing plus CO breathing, significant increases were found in mRNA levels for nuclear respiratory factor-1, peroxisome proliferator-activated receptor-γ co- activator-1 aα mitochondrial transcription factor-A (Tfam), and DNA polymerase -γ (Polγ) with no change in mitochondnal DNA (mtDNA) copy number or VO2max. Levels of myosin heavy chain I and nuclear- encoded HO-1, superoxide dismutase-2, citrate synthase, mitofusin- and -2, and mitochondrial-encoded cytochrome oxidase subunit-1(COX-I) and ATPase-6 proteins increased significantly. None of these responses were reproduced by VO2max testing alone, whereas CO alone increased Tfam and Pol-γ mRNA, and COX-I, ATPase-6, mitofusin-2, HO-1, and superoxide dismutase protein. These findings provide evidence linking the HO/CO response involved in mitochondrial biogenesis in rodents to skeletal muscle in humans through a set of responses involving regulation of the mtDNA transcriptosome and mitochondrial fusion proteins autonomously of changes in exercise capacity. [ABSTRACT FROM AUTHOR]

In a previous study we demonstrated that the institution of biventricular pacing in chronic heart failure (CHF) acutely facilitates the arterial baroreflex. The arterial baroreflex has important prognostic value in CHF. We hypothesized that the acute response in baroreflex sensitivity (BRS) after the institution of cardiac resynchronization therapy (CRT) has predictive value for midterm response. One day after implantation of a CRT device in 33 CHF patients (27 male/6 female; age, 66.5 ± 9.5 yr; left ventricular ejection fraction, 28 ± 7%) we measured noninvasive BRS and heart rate variability (HRV) in two conditions: CRT device switched on and switched off (on/off order randomized). Echocardiography was performed before implantation (baseline) and 6 mo after implantation (follow-up). CRT responders were defined as patients in whom left ventricular end-systolic volume at follow-up had decreased by ⩾15%. Responders (69.7%) and nonresponders (30.3%) had similar baseline characteristics. In responders, CRT increased BRS by 30% (P = 0.03); this differed significantly (P = 0.02) from the average BRS change (-2%) in the nonresponders. CRT also increased HRV by 30% in responders (P = 0.02), but there was no significant difference found compared with the increase in HRV (8%) in the nonresponders. Receiver-operating characteristic curve analysis revealed that the percent BRS increase had predictive value for the discrimination of responders and nonresponders (area under the curve, 0.69; 95% confidence interval, 0.5 1- 0.87; maximal accuracy, 0.70). Our study demonstrates that a CRT-induced acute BRS increase has predictive value for the echocardiographic response to CRT. This finding suggests that the autonomic nervous system is actively involved in CRT-related reverse remodeling. [ABSTRACT FROM AUTHOR]

The present study tested the hypothesis that short-term dietary deficiency of magnesium (Mg) (21 days) in rats would 1) result in decreased serum(s) [the present study tested the levels of Mg, sphingomyelin (SM), and phosphatidylcholine (PC)]; 2) promote DNA fragmentation, lipid peroxidation (LP), and activation of caspase-3 in cardiac (ventricular and atrial) and vascular(aortic) muscle; and 3) low levels of Mg2+ added to drinking water would either prevent or greatly ameliorate these manifestations. The data indicate that short-term Mg deficiency (10% normal dietary intake) resulted in profound reductions in serum-ionized Mg and total Mg with an elevation in serum-ionized calcium (Ca2+), significant lowering of serum SM and serum PC, with concomitant LP, DNA fragmentation, and activation of caspase-3 in ventricular (right and left chambers), atrial (right and left chambers) and abdominal aortic smooth muscle. The greater the reduction in serum-ionized Mg, the greater the effects on DNA fragmentation, LP, and caspase-3 activity. The intake of water-borne Mg2+ at all levels greatly attenuated or inhibited the reductions in serum SM and serum PC, activation of LP, DNA fragmentation, and the activation of caspase-3; even very low levels of Mg2+ in drinking water (i.e., 15 parts·million-1 day-1) were cardio and vascular protective. In addition, we demonstrate that short-term dietary deficiency of Mg probably results in a downregulation of SM synthase and a decreased synthesis of PC. [ABSTRACT FROM AUTHOR]

Falção-Pires I, Gonçalves N, Henriques-Coelho T, MoreiraGonçalves D, Roncon-Albuquerque R Jr, Leite-Moreira AF. Apeun decreases myocardial injury and improves right ventricular function in monocrotaline-induced pulmonary hypertension. Am I Physiol Heart Circ Physiol 296: H2007-H20l4, 2009. First published April 3, 2009; doi: 10.11 52/ajpheart.00089.2009.-We investigated the endogenous production of apelin and the cardiac and pulmonary effects of its chronic administration in monocrotaline (MCT)-induced pulmonary hypertension (PH). Male Wistar rats were injected with MCT (60 mg/kg sc) or vehicle (day 0). One week later, these animals were randomly treated during 17 days with pyroglutamylated apelin-13 (Pyr-API3; 200 μg∙kg[sup-1]day[sup-1] ip) or a similar volume of saline, resulting in four groups: sham (n = 11), sham-AP (a = 11), MCT (n = 16), and MCT-AP (a = 13). On day 25, right ventricular (RV) and left ventricular (LV) hemodynamic and morphometric parameters were assessed. Tissue and plasma samples were collected for histological and molecular analysis. When compared with sham, the MCT group presented a significant increase of RV mass (166 ± 38%), diameter of cardiomyocyte (40 ± 10%), myocardial fibrosis (95 ± 20%), peak systolic pressure (99 ± 22%), peak rate of ventricular pressure rise (dP/dt[submax] 74 ± 24%), peak rate of ventricular pressure decline (dP/dt[submin] 73 ± 19%), and time constant T (55 ± 16%). In these animals, RV expression of apelin (-73 ± 10%) and its receptor APJ (-61 ± 20%) was downregulated, whereas mRNA expression of type B natriuretic peptide (9,606 ± 713%), angiotensinogen (191 ± 147%), endothelin-l (RV, 497 ± 156%; and LV, 799 ± 309%), plasmatic levels of apelin (104 ± 48%), and angiotensin 1-7 (161 ± 151 %) were increased. Chronic treatment with Pyr-AP 13 significantly attenuated or normalized these changes, preventing apelin-APJ mRNA downregulation and PH-induced neurohumoral activation of several vasoconstrictors, which exacerbates apelin-APJ vasodilator effects. Therefore, apelin delayed the progression of RV hypertrophy and diastolic dysfunction. Together, these observations suggest that the apelin-APJ system may play an important role in the pathophysiology of PH, representing a potential therapeutic target since it significantly attenuates RV overload and PH-induced neurohumoral activation. [ABSTRACT FROM AUTHOR]

Lund DD Chu Y, Miller JD, Heistad DD. Protective effect of extracellular superoxide dismutase on endothelial function during aging. Am J physiol Heart Circ Physiol 296: H1920-H1925, 2009. First published April 17, 2009; doi:10.1152/ajpheart.01342.2008.-Endothelial vasomotor function decreases with increasing age. Extracellular superoxide dismutase (ecSOD) protects against vascular dysfunction in several disease states. The purpose of this study was to determine whether endogenous ecSOD protects against endothelial dysfunction ip old mice. Vasomotor function of the aorta was studied ex vivo in wild-type (ecSOD[sup+/+]) and ecSOD-deficient (ecSOD[sup-/-]) mice at 11 (adult) and 29 (old) mo of age. Maximal relaxation to acetylcholine (10[sup-4] M) was impaired in vessels from adult ecSOD mice [75 ± (mean ± SE)1 compared with wild-type mice (89 ± 2%, P < 0.05). Maximal relaxation to acetylcholine (10[sup-4] M) was profoundly impaired in aorta from old ecSOD[sup-/-] mice (45 ± 5%) compared with wild-type mice (75 ± 4%, P < 0.05). There was a significant correlation between expression of ecSOD and maximal relaxation to acetylcholine in adult and old mice. Tempol (1 mM), a scavenger of uperoxide, improved relaxation in response to acetylcholine (63 8%) in old ecSOD[sup-/-] mice (P < 0.05), but not wild-type mice (75 ± 4%). Maximal relaxation to sodium nitroprusside was similar in aorta from adult and old wild-type and ecSOD[sup-/-] mice. Quantitative RT-PCR showed a decrease in mRNA levels of ecSOD and catalase in aorta of old mice and an increase in levels of TNFα and Nox-4 in aorta of old mice compared with adult mice. The findings support the hypothesis that impaired antioxidant mechanisms may contribute to cumulative increases in oxidative stress and impaired endothelial function in old mice. In conclusion, endogenous ecSOD plays an important role in protection against endothelial dysfunction during aging. [ABSTRACT FROM AUTHOR]

Rosenfeld CR, Liu X, DeSpain K. Pregnancy modifies the large conductance qa2±-activated K channel and cGMP-dependent signaling pathway in uterine vascular smooth muscle. Am J Physiol Heart Circ Physiol 296: H1878-H1887, 2009; doi:10.1152/ajpheart.01185.2008.Regulation of uteroplacental blood flow (UPBF) during pregnancy remains unclear. Large conductance, Ca[sup2+]-activated K[sup+] channels (BK[subCa]), consisting of aand regulatory 13-subunits, are expressed in uterine vascular smooth muscle (UVSM) and contribute to the maintenance of UPBF in the last third of ovine pregnancy, but their expression pattern and activation pathways are unclear. We examined BKca subunit expression, the cGMP-dependent signaling pathway, and the functional role of BKca in uterine arteries (UA) from nonpregnant (n 7), pregnant (n = 38; 56-145 days gestation; term, -150 days), and postpartum (n = 15; 2-56 days) sheep. The a-subunit protein switched from 83-87 and 105 kDa forms in nonpregnant UVSM to 190 kDa throughout pregnancy, reversal occurring >30 days postpartum. The 39-kDa 13,-subunit was the primary regulatory subunit. Levels of 100-kDa a-subunit rose -70% during placentation (P < 0.05) and were unchanged in the last two-thirds of pregnancy; in contrast, β[sub1]-protein rose throughout pregnancy (R[sub2] = 0.996; P < 0.001; n = 13), increasing 50% during placentation and approximately twofold in the remainder of gestation. Although UVSM soluble guanylyl cyclase was unchanged, cGMP and protein kinase G[sub1α] increasecd (P < 0.02), paralleling the rise and fall in β[sub1]-protein during pregnancy and the puerperium. BK[subca] inhibition not only decreased UA nitric oxide (NO)-induced relaxation but also enhanced a-agonist-induced vasoconstriction. UVSM BK[subCa] modify relaxationcontraction responses in the last two-thirds of ovine pregnancy, and this is associated with alterations in α-subunit composition, α:β,subunit stoichiometry, and upregulation of the cGMP-dependent pathway, suggesting that BK[subca] activation via NO-cGMP and β[sub1] augmentation may contribute to the regulation of UPBF. [ABSTRACT FROM AUTHOR]

Peluffo G, Calcerrada P, Piacenza L, Pizzano N, Radi R. Superoxidemediated inactivation of nitric oxide and peroxynitrite formation by tobacco smoke in vascular endothelium: studies in cultured cells and smokers. Am J P/iysiol Heart Circ Physiol 296: H 1781-H 1792, 2009. First published April 10, 2009; doi:10.1152/ajpheart.00930.2008.-Tobacco smoke is known to cause nitric oxide (∙NO) inactivation and endothelial dysfunction. In this work we evaluated the interplay between [sup∙]NO and superoxide (Or) radicals and the consequent impact on [sup&2219;]NO bioavailability and nitroxidative stress in bovine aortic endothelial cells exposed to cigarette smoke extract (CSE) and in smokers. Bovine aortic endothelial cells in the presence of CSE triggered [sub2][sup∙-] production as indicated by spin-trapping electron paramagnetic resonance experiments. 0 was produced both extracellulary (3.4 vs. 1.0 nmol∙h[sup-1]∙mg[sup-1] CSE vs. control; cytochrome c[sup3+] reduction assay) and intracellularly (40% inhibition of cytosolic aconitase). CSE also led to the production of peroxynitrite as evaluated by dihydrorhodamme oxidation and protein tyrosine nitration on cells. O[sub2][sup∙-] and peroxynitrite formation were decreased by ascorbate and a-tocopherol. Additionally, CSE led to the oxidation of endothelial nitric oxide synthase increasing the monomeric inactive form of endothelial nitric oxide synthase. Smokers and age-matched healthy volunteers were supplemented orally with 500 mg ascorbate plus 400 IU all-rac-citocopherol every 12 h for 165 days. Smokers had endothelial dysfunction compared with control subjects (95% confidence interval: 2.5, 8.3 vs. 10.6, 14.2; P < 0.05) as assessed by flow-mediated dilation of the brachial artery, and plasma levels of protein 3-nitrotyrosine were 1.4-fold higher. The loss of flow-mediated dilation in smokers reverted after a long-term antioxidant supplementation (95% confidence interval: 13.9, 19.9; P < 0.05), reaching values comparable with the control population. Our data indicate that elements on tobacco smoke, most likely through redox cycling, divert NO toward peroxynitrite by inducing O[sub2][sup∙-] production in vascular endothelial cells both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Itoh A, Krishnamurthy G, Swanson JC, Ennis DB, Bothe W, KuhI E, Karlsson M, Davis LR, Miller DC, Ingels NB Jr. Active stiffening of mitral valve leaflets in the beating heart. Am J Physiol Heart Circ Jrhysiol 296: H1766-H1773, 2009. First published April 10, 2009; doi:10.1 152/ajpheart.00120.2009.-The anterior leaflet of the mitral valve (MV), viewed traditionally as a passive membrane; is shown to be a highly active structure in the beating heart. Two types of leaflet contractile activity are demonstrated: 1) a brief twitch at the beginning of each beat (reflecting contraction of myocytes in the leaflet in communication with and excited by left atrial muscle) that is relaxed by midsystole and whose contractile activity is eliminated with β-receptor blockade and 2) sustained tone during isovolumic relaxation, insensitive to p-blockade, but doubled by stimulation of the neurally rich region of aortic-mitral continuity. These findings raise the possibility that these leaflets are neurally controlled tissues, with potentially adaptive capabilities to meet the changing physiological demands on the heart. They also provide a basis for a permanent paradigm shift from one viewing the leaflets as passive flaps to one viewing them as active tissues whose complex function and dysfunction must be taken into account when considering not only therapeutic approaches to MV disease, but even the definitions of MV disease itself. [ABSTRACT FROM AUTHOR]

Manzi V, Castagna C, Padua E, Lombardo M, D'Ottavio S, Massaro M, Volterrani M, lellamo F. Dose-response relationship of autonomic nervous system responses to individualized training impulse in marathon runners. Am J Physiol Heart Circ Physiol 296: H1733-H1740, 2009. First published March 27, 2009; doi: 10.11 52/ajpheart.00054.2009.-In athletes, exercise training induces autonomic nervous system (ANS) adaptations that could be used to monitor training status. However, the relationship between training and ANS in athletes has been investigated without regard for individual training loads. We tested the hypothesis that in longdistance athletes, changes in ANS parameters are dose-response related to individual volume/intensity training load and could predict athletic performance. A spectral analysis of heart rate (HR), systolic arterial pressure variability, and baroreflex sensitivity by the sequences technique was investigated in eight recreational athletes during a 6-mo training period culminating with a marathon. Individualized training load responses were monitored by a modified training impulse (TRIMP1) method, which was determined in each athlete using the individual HR and lactate profiling determined during a treadmill test. Monthly TRIMP1 steadily increased during the training period. All the ANS parameters were significantly and very highly correlated to the dose of exercise with a second-order regression model (r[sup2] ranged from 0.90 to 0.99; P < 0.001). Variance, high-frequency oscillations of HR variability (HRV), and baroreflex sensitivity resembled a bell-shaped curve with a minimum at the highest TRIMP,, whereas low-frequency oscillations of HR and systolic arterial pressure variability and the low frequency (LF)-to-high frequency ratio resembled an U-shaped curve with a maximum at the highest TRIMP1. The LF component of HRV assessed at the last recording session was significantly and inversely correlated to the time needed to complete the nearing marathon. These results suggest that in recreational athletes, ANS adaptations to exercise training are dose related on an individual basis, showing a progressive shift toward a sympathetic predominance, and that LF oscillations in HRV at peak training load could predict athletic achievement in this athlete population. [ABSTRACT FROM AUTHOR]

Stauss HM, Rarick KR, Deklotz RJ, Sheriff DD. Frequency response characteristics of whole body autoregulation of blood flow in rats. Am J Physiol Heart Circ Physiol 296: HI 607-HI 616, 2009. First published February 27, 2009; doi: 10.11 52/ajphearrt.0 1262.2008.Previously, we demonstrated that very low-frequency (VLF) blood pressure variability (BPV) depends on voltage-gated L-type Ca[sup2+]channels, suggesting that autoregulation of blood flow and/or myogenic vascular function significantly contributes to VLF BPV. To further substantiate this possibility, we tested the hypothesis that the frequency response characteristic of whole body autoregulation of blood flow is consistent with the frequency range of VLF BPV (0.02-0.2 Hz) in rats. In anesthetized rats (n = 11), BPV (0.0 16-0.5 Hz) was induced by computer-regulated cardiac pacing while blood pressure, heart rate, and cardiac output (CO) were recorded during control conditions (NaCl, I mI/h iv) and during ai-adrenergic receptor stimulation (phenylephrine, 1 mg∙ml[sup-1]∙h[sup-1] iv) that has been reported to facilitate myogenic vascular function. Baroreceptor-heart rate reflex responses were elicited to confirm a functional baroreflex despite anesthesia. During control conditions, transfer function analyses between mean arterial pressure (MAP) and GO, and between MAP and total vascular conductance (CO/MAP) indicated autoregulation of blood flow at 0.0 16 Hz, passive vascular responses between 0.033 and 0.2 Hz, and vascular responses compatible with baroreflexmediated mechanisms at 0.333 and 0.5 Hz. Stimulation of α[sub1]-adrenergic receptors extended the frequency range of autoregulation of blood flow to frequencies up to 0.033 Hz. In conclusibn, depending on sympathetic vascular tone, whole body autoregulati9n of blood flow operates most effectively at frequencies below 0.05 Hz. This frequency range overlaps with the lower end of the frequency band of VLF BPV in rats. Baroreceptor reflex-like mechanisms contribute to LF (0.2-0.6 Hz) but not VLF BPV-induced vascular responses. [ABSTRACT FROM AUTHOR]

Meyer KD, Zhang H, Zhang L Prenatal cocaine exposure abolished ischemic preconditioning-induced protection in adult male mt hearts: role of PKCϵ. Am J Physiol Heart Circ Physiol 296: H 1566-Hi 576, 2009. First published March 13, 2009; doi:10.1 l52/ajpheart.00898.2008.-Prenatal cocaine exposure in rats resulted in decreased PKCϵ protein expression in the heart of adult male but not female offspring. The present study determined its functional consequence of ,inhibiting cardioprotection mediated by ischemic preconditioning. Pregnant Sprague-Dawley rats were administered intraperitoneally saline or cocaine (30 mg∙kg[sup-1]∙day[sup-1]) from day 15 to day 21 of gestational age. Hearts were isolated from 3-mo-old offspring and were subjected to ischemia and reperfusion injury in a Langendorff preparation, with or without prior ischemic preconditioning. Preischemic values of left ventricular function were the same between the saline control and cocaine-treated animals. Ischemic preconditioning of two episodes of 5-mm ischemia significantly decreased infarct size and enhanced postischemic functional recovery of the left ventricle in the saline control animals. This ischemic preconditioning was associated with increased phospho-PKCϵ, but not phospho-PKCϵ, levels and was blocked by a PKCϵ translocation inhibitor peptide. Prenatal cocaine treatment abolished the ischemic preconditioning-mediated increase in phospho-PKCϵ and cardioprotection in the heart of male offspring. In contrast, the cardioprotective effect was fully maintained in female offspring that were exposed to cocaine before birth. The results suggest that prenatal cocaine exposure causes a sex-specific loss of cardioprotection by ischemic preconditioning in adult offspring, which is most likely due to fetal programming of PKCϵ gene repression, resulting in a downregulation of PKCϵ function in the heart of adult male offspring. [ABSTRACT FROM AUTHOR]

The adenosine A2A receptor (A2AR) enhances cardiac contractility, and the adeno- sine A1R receptor (A1R) is antiadrenergic by reducing the adrenergic ~ receptor (I3iR)-elicited increase in contractility. In this study we compared the A2AR-, A1R-, and 131R-elicited actions on isolated rat ventricular myocytes in terms of Ca transient and contractile re- sponses involving PKA and PKC. Stimulation of A2AR with 2 p~M (-.~EC50) CGS-21680 (CGS) produced a 17-28% increase in the Ca transient ratio (CTR) and maximum velocities (Vm~,,~) of transient ratio increase (+MVT) and recovery (-MVT) but no change in the time-to-50% recovery (TTR). CGS increased myocyte sarcomere shortening (MSS) and the maximum velocities of shortening (+MVS) and relaxation (-MVS) by 31-34% with no change in time-to-50% relengthening (TTL). ~1R stimulation using 2 nM (-~EC50) isopro- terenol (Iso) increased CTR, +MVT, and -MVT by 67-162% and decreased TTR by 43%. Iso increased MSS, +MVS, and -MVS by 153-174% and decreased TI'L by 31%. The A2AR and ~31R Ca transient and contractile responses were not additive. The PKA inhibitor Rp-adenosine 3' ,5 `-cyclic monophosphorothioate triethyla- monium salt prevented both the CGS- and Iso-elicited contractile responses. The PKC inhibitors chelerythrine and KJE1-l peptide (PKCe specific) prevented the antiadrenergic action of A1R but did not influence A2AR-mediated increases in contractile variables. The findings suggest that cardiac A2AR utilize cAMPIPKA like 31R, but the Ca transient and contractile responses are less in magnitude and not equally affected. Although PKC is important in the ~ R antiad- renergic action, it does not seem to play a role in A2AR-elicited Ca transient and contractile events. [ABSTRACT FROM AUTHOR]

Andresen MC, Peters JH. Comparison of baroreceptive to other afferent synaptic transmission to the medial solitary tract nucleus. Am J Physiol Heart Circ Physiol 295: H2032-H2042, 2008. First published September 12, 2008; doi: 10.1152/ajpheart.00568.2008.Cranial nerve visceral afferents enter the brain stem to synapse on neurons within the solitary tract nucleus (NTS). The broad heterogeneity of both visceral afferents and NTS neurons makes understanding afferent synaptic transmission particularly challenging. To study a specific subgroup of second-order neurons in medial NTS, we anterogradely labeled arterial baroreceptor afferents of the aortic depressor nerve (ADN) with lipophilic fluorescent tracer (i.e., ADN+) and measured synaptic responses to solitary tract (ST) activation recorded from dye-identified neurons in medial NTS in horizontal brain stem slices. Every ADN+ NTS neuron received constant-latency ST evoked excitatory postsynaptic currents (EPSC5) (jitter <192 μs, SD of latency). Stimulus-recruitment profiles showed single thresholds and no suprathreshold recruitment, findings consistent with EPSCs arising from a single, branched afferent axon. Frequency-dependent depression of ADN+ EPSCs averaged ∼70% for five shocks at 50 Hz, but single-shock failure rates did not exceed 4%. Whether adjacent ADNor those from unlabeled animals, other second-order NTS neurons (jitters <200 μs) had ST transmission properties indistinguishable from ADN+. Capsaicin (CAP; 100 nM) blocked ST transmission in some neurons. CAP-sensitive ST-EPSCs were smaller and failed over five times more frequently than CAP-resistant responses, whether ADN+ or from unlabeled animals. Variance-mean analysis of ST-EPSCs suggested uniformly high probabilities for quantal glutamate release across second-order neurons. While amplitude differences may reflect different numbers of contacts, higher frequencydependent failure rates in CAP-sensitive ST-EPSCs may arise from subtype-specific differences in afferent axon properties. Thus afferent transmission within medial NTS differed by axon class (e.g., CAP sensitive) but was indistinguishable by source of axon (e.g., baroreceptor vs. nonbaroreceptor). [ABSTRACT FROM AUTHOR]

von der Weid PY, Rahman M, Imtiaz MS, van Helden DF. Spontaneous transient depolarizations in lymphatic vessels of the guinea pig mesentery: pharmacology and implication for spontaneous contractility. Am J Physiol Heart Circ Physiol 295: H1989-H2000, 2008; doi: 10.1152/ajpheart.00007.2008.-Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca[sup2+] imaging, and pharmacological agents. In our investigation of the role of intracellular Ca[sup2+] released from Ca[sup2+] stores, we observed that intracellular Ca[sup2+] transients accompanied some STDs, although there were many exceptions where Ca[sup2+] transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP3Rs) but not by treatments known to alter Ca[sup2+] release via ryanodine receptors. A role for Ca[sup2+]-activated Cl[sup-] (Cl[subCa]) channels was indicated, as STDs were dependent on the Cl[sup-] but not Na[sup+] concentration of the superfusing solution and were inhibited by the Cl[subCa] channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl[sup-] blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cationlstoreoperated channel blockers SKF-96365, Gd[sup3+] and Ni[sup2+] had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd[sup3+], and Ni[sup2+] altered the occurrence of spontaneous APs. Our findings support a role for Ca[sup2+] release through IP3Rs and a resultant opening of Cica channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking. [ABSTRACT FROM AUTHOR]

Miyamoto T, Kawada T, Yanagiya Y, Akiyama T, Kamiya A, Mizuno M, Takaki H, Sunagawa K, Sugimachi M. Contrasting effects of presynaptic α2-adrenergic autoinhibition and pharmacologic augmentation of presynaptic inhibition on sympathetic heart rate control. Am J Physiol Heart Circ Physiol 295: H1855-H1866, 2008. First published August 29, 2008; doi: 10.1152/ajpheart.522.2008.-Presynaptic α2-adrenergic receptors are known to exert feedback inhibition on norepinephrine release from the sympathetic nerve terminals. To elucidate the dynamic characteristics of the inhibition, we stimulated the right cardiac sympathetic nerve according to a binary white noise signal while measuring heart rate (HR) in anesthetized rabbits (n = 6). We estimated the transfer function from cardiac sympathetic nerve stimulation to HR and the corresponding step response of HR, with and without the blockade of presynaptic inhibition by yohimbine (1 mg/kg followed by 0.1 mg∙kg[sup-1]∙h[sup-1] iv). We also examined the effect of the α2-adrenergic receptor agonist clonidine (0.3 and 1.5 mg∙kg[sup-1]∙h[sup-1] iv) in different rabbits (n = 5). Yohimbine increased the maximum step response (from 7.2 ± 0.8 to 12.2 ± 1.7 beats/mm, means ± SE, P < 0.05) without significantly affecting the initial slope (0.93 ± 0.23 vs. 0.94 ± 0.22 beats∙min[sup-1] s[sup-1]). Higher dose but not lower dose clonidine significantly decreased the maximum step response (from 6.3 ± 0.8 to 6.8 ± 1.0 and 2.8 ± 0.5 beats/mm, P < 0.05) and also reduced the initial slope (from 0.56 ± 0.07 to 0.51 ± 0.04 and 0.22 ± 0.06 beats∙min[sup-1]s[sup-1], P < 0.05). Our findings indicate that presynaptic α2-adrenergic autoinhibition limits the maximum response without significantly compromising the rapidity of effector response. In contrast, pharmacologic augmentation of the presynaptic inhibition not only attenuates the maximum response but also results in a sluggish effector response. [ABSTRACT FROM AUTHOR]

Right ventricular (RV) dysfunction is a common long-term complication in patients after the repair of congenital heart disease. Previous investigators have examined the cellular and molecular mechanisms of left ventricular (LV) remodeling, but little is known about the stressed RV. Our purpose was to provide a detailed physiological characterization of a model of RV hypertrophy and failure, including RV-LV interaction, and to compare gene alterations between afterloaded RV versus LV. Pulmonary artery constriction was performed in 86 mice. Mice with mild and moderate pulmonary stenosis (PS) developed stable hypertrophy without decompensation. Mice with severe PS developed edema, decreased RV function, and high mortality. Tissue Doppler imaging demonstrated septal dyssynchrony and deleterious RV-LV interaction in the severe PS group. Microarray analysis showed 196 genes with increased expression and 1,114 with decreased expression. Several transcripts were differentially increased in the afterloaded RV but not in the afterloaded LV, including clusterin, neuroblastoma suppression of tumorigenicity 1, Dkk3, Sfrp2, formin binding protein, annexin A7, and lysyl oxidase. We have characterized a murine model of RV hypertrophy and failure, providing a platform for studying the physiological and molecular events of RV remodeling. Although the molecular responses of the RV and LV to afterload stress are mostly concordant, there are several key differences, which may represent targets for RV failure-specific therapy. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the role of hydrogen sulphide (H2S) in the cardioprotection induced by ischemic postconditioning and to examine the underlying mechanisms. Cardiodynamics and myocardial infarction were measured in isolated rat hearts. Postconditioning with six episodes of 10-s ischemia (IPostC) significantly improved cardiodynamic function, which was attenuated by the blockade of endogenous H2S production with D-L-propargylglycine. Moreover, IPostC significantly stimulated H2S synthesis enzyme activity during the early period of reperfusion. However, D-L-propargylglycine only attenuated the IPostC-induced activation of PKC-α and PKC-ε but not that of PKC-δ, Akt, and endothelial nitric oxide synthase (eNOS). These data suggest that endogenous H2S contributes partially to the cardioprotection of IPostC via stimulating PKC-α and PKC-ε. Postconditioning with six episodes of a 10-s infusion of NaHS (SPostC) or 2 min continuous Na}IS infusion (SPostC2) stimulated activities of Akt and PKC, improved the cardiodynamic performances, and reduced myocardial infarct size. The blockade of Akt with LY-294002 (15 nM) or PKC with chelerythrine (10 µM) abolished the cardioprotection induced by H2S postconditioning. SPostC2, but not SPostC, also additionally stimulated eNOS. We conclude that endogenous H2S contributes to IPostC-induced cardioprotection. H2S postconditioning confers the protective effects against ischemia-reperfusion injury through the activation of Akt, PKC, and eNOS pathways. [ABSTRACT FROM AUTHOR]

PKG activator 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (CPT) at reperfusion protects ischemic hearts, but the mechanism is unknown. We recently proposed that in preconditioned hearts PKC lowers the threshold for adenosine to initiate signaling from low-affinity A2b receptors during early reperfusion thus allowing endogenous adenosine to activate survival kinases phosphatidylinositol 3-kinase (PI3K) and ERK. We tested whether CPT might also sensitize A2b receptors to adenosine. CPT (10 µM) during the first minutes of reperfusion markedly reduced infarction in isolated rabbit hearts undergoing 30-min regional ischemia/2-h reperfusion, and salvage was blocked by MRS 1754, an A2b-selective antagonist. Coadministration of wortmannin (PI3K inhibitor) or PD-98059 (MEK1/2 and therefore ERK1/2 inhibitor) also blocked protection. In nonischemic hearts, 10-min infusion of CPT did not change phosphorylation of Akt or ERK1/2. Neither did a subthreshold dose (2.5 nM) of the nonselective but A2b-potent receptor agonist 5′-(N-ethylcarboxamido)adenosine (NECA). However, when 2.5 nM NECA was combined with 10 µM CPT, both phospho-Akt and phospho-ERK1/2 significantly increased, indicating CPT had lowered the threshold for Am-dependent signaling. The PKC antagonist chelerythrine blocked this phosphorylation induced by CPT + NECA. Chelerythrine also blocked the anti-infarct effect of CPT as did nonselective (glibenclamide) and mitochondrial-selective (5-hydroxydecanoate) KATP channel blockers. A free radical scavenger, N-(2-mercaptopropionyl)glycine, also blocked CPT protection. We propose CPT targets PKG, which activates PKC through mitochondrial KATP channel (mitoKATP)-dependent redox signaling, a sequence mimicking that already documented in preconditioning. Activated PKC then augments sensitivity of normally low-affinity cardiac adenosine A2b receptors so endogenous adenosine can protect by activating Akt and ERK. [ABSTRACT FROM AUTHOR]